Abstract/Summary Rationale: Role of protein kinases in cell functions including growth, differentiation, and cell death is well established. Phosphorylation of proteins by cognate protein kinases is a dynamic process known to profoundly influence the physiological function of proteins in the cell. This process can become dysregulated in disease states including in cancer, and attempts have been made to target certain protein kinases for therapy of breast cancer (and other cancers). The focus of this laboratory is to define the physiological functions of a novel member of a protein kinase family, called Mixed Lineage Kinases (MLKs). MLK family members are unique in the sense that they can target both Ser/Thr and tyrosine residues for phosphorylation. The function of this novel family of kinases in tumorigenesis is not well known. We have made the original observation that MLK3 directly interacts with and phosphorylates a protein called Pin1. Pin1 is known to be highly expressed in breast tumors and promotes survival and proliferation of breast cancer cells. Pin1 is a member of peptidyl prolyl cis/trans isomerase family, which binds to its substrates and isomerizes specific pSer/Thr-Pro motifs and catalytically induces conformational changes. Such conformational changes can have profound effects on the role of Pin1 substrates so as to significantly impact cellular functions. However, the mechanism by which Pin1 promotes breast cancer pathogenesis is unclear. Our observation that Pin1 is directly phosphorylated by MLK3 at a very specific residue resulting in the activation of its isomerase activities (a hall mark of increased Pin1 function) may be important in its tumorigenic function. This is supported by our observation that phospho-Pin1 is exclusively localized in the nucleus of a highly aggressive breast cancer cell line and promotes G2/M transition of cell cycle. These observations suggest that phosphorylated Pin1 might have functional target(s) in the nucleus to promote cell cycle progression. Since several targets of Pin1 exist in the nucleus and may contribute to the oncogenic process, our results would contribute to this knowledge by specifically identifying targets of the phospho-Pin1 in the nucleus and their potential role in breast cancer. Hypothesis: Based on our novel finding that Pin1 is directly phosphorylated and activated by MLK3, we hypothesize that phosphorylated Pin1 targets specific substrate(s) in the nucleus which may serve to promote breast cancer pathogenesis. Objectives: To address the above hypothesis, we propose three Specific Aims: (1) To characterize the phosphorylation of Pin1 and identify the target(s)/substrate(s) of phospho-Pin1. (2) To determine the functional consequences of Pin1 phosphorylation (by MLK3) in breast cancer pathogenesis. (3) To determine whether pharmacological or genetic disruption of MLK3 inhibits the pathological functions of Pin1 in breast cancer pathogenesis. Procedures to be used: These studies will employ a broad range of technical approaches, including gene silencing by specific RNAi/ShRNA, immunoprecipitation, kinase assays, Western blotting, cell proliferation and cell death assays. Study models will include cultured cells and xenografts of breast cancer in mice. Significance of potential new findings: The results of these studies will elucidate, for the first time, detailed mechanism by which MLK3 regulates Pin1 phosphorylation and modulates its function during breast cancer pathogenesis. Further, our results will point to the possibility of utilizing MLK inhibitor(s) as a novel therapeutic intervention for breast cancer therapy. PUBLIC HEALTH RELEVANCE: Project Narrative As more women enter the military service, there is going to be an increasing number of female veterans who may be impacted by the incidence of breast cancer. The established therapies for breast cancer include surgery frequently augmented by radiation and/or chemotherapy. However, the failure rate of therapy, and recurrence of the disease following remission continues to remain a challenge. Thus there is a critical need for novel therapeutic approaches especially targeted to specific signals in the tumor cells. Protein kinases have been considered as among the most important targets for therapy of cancer and other diseases. We have discovered that protein kinase MLK3 is highly active in breast tumors and is involved in the activation of another protein Pin1 which has previously been implicated in breast cancer pathogenesis. Thus, we propose to investigate the mechanism by which MLK3 regulates Pin1, and to examine the application of MLK inhibitors as potential therapeutic approach for breast cancer treatment. These studies accord with the mission of the V.A. Med. Res.